Throughout this application various publications are referenced by arabic numbers within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
Cellular oncogenes are normal genes which have been conserved throughout evolution and are believed to have normal functional roles in the cell. In their non-activated (wild-type) state such cellular oncogenes are sometimes referred to as proto-oncogenes. Proto-oncogenes are not oncogenic or tumorigenic until they are activated in some way. A number of different genetic mechanisms may cause the somatic mutation of oncogenes that results in the activated oncogenes found in tumor cells. These include point mutations, translocations, gene rearrangement, and gene amplification, all of which may be induced by chemical or physical carcinogenic means or by the integration of a viral genome adjacent to the proto-oncogene sequences in the host DNA. Certain oncogenes, such as ras and wild type p53 oncogenes, when "activated" encode mutant proteins while others such as myc may express elevated levels of normal protein.
The wild type p53 oncogene encodes wild type p53 polypeptide which functions as a negative regulator of cell division. The wild type p53 polypeptide has been found intracellularly in normal cells and tissues at low levels. Mutant p53 polypeptides encoded by activated p53 oncogenes are present intracellularly at high concentrations in mammalian tumors and tumor cell lines.
The wild type p53 oncogene is conserved across a wide variety of species including man, mouse, rat and frog (1). cDNA sequence analysis has indicated that there are five blocks of very highly conserved sequences (2, 3). These conserved residues have been grouped in blocks beginning at amino acid 117 and ending at amino acid 286 (4). Point mutations, occurring principally in these five blocks of very highly conserved sequences and also in highly conserved regions of the wild type p53 oncogene lying outside of these blocks, produce an activated p53 oncogene (SEQ ID NO:2-SEQ ID NO:7). Changes in these conserved areas have a significant impact on the function of the mutant p53 polypeptide. Changes in these regions of the wild type p53 oncogene generate an activated p53 oncogene which encodes a protein having a conformational change identical to the vast majority of the mutant p53 polypeptides so expressed.
The product of the activated p53 oncogene, i.e. mutant p53 polypeptide, is present at high levels in a high percentage of virtually all classes of human tumors including tumors of the colon, lung, and breast (2). Biochemical analyses of mutant p53 polypeptides demonstrate that activating mutations affect the polypeptide's structure in similar ways. Mutant p53 polypeptides have a much longer half-life as compared to normal p53 polypeptide. In addition, mutant p53 polypeptides are able to complex with the heat-shock-protein-70 family of proteins but not to SV40 large T antigen (5-8). Finally, in histological or cell-based assays, mutant and wild-type p53 polypeptides have been distinguished on the basis of differential reactivity with monoclonal antibodies. Antibody secreted by clone PAb246 is reactive with wild-type p53 polypeptides but not with any mutant p53 polypeptide tested to date, while antibody secreted by clone PAb240 reacts with all mutant p53 polypeptides tested to date but not with a wild-type p53 polypeptide (5-21).
The human, wild-type p53 oncogene is found on chromosome 17p. Allelic loss in 17p occur at high frequency in human breast cancer (22), colon cancer (23), astrocytomas (24) and small cell lung carcinoma (25). The question of allele loss of the wild-type p53 oncogene was addressed by cytogenetic analysis of a number of colon cancer samples using specific DNA probes (23). The results of such analysis indicated that at least a portion, if not all, of one of the two alleles of the wild-type p53 oncogene was lost. However, there were no large deletions or rearrangements in the p53 oncogene associated with the other allele. In most cases, sequence analysis of cDNA from the tumors demonstrated that the p53 oncogene encoded by the second allele contained activating point or missense mutations in those regions encoding the conserved amino acid sequence boxes (26). Mutant p53 polypeptides possess an extended half-life of about 24 hours in comparison to wild-type p53 polypeptides which have a half-life of about 20 minutes. The extended half-life of the mutant p53 polypeptide allows it to accumulate to detectable levels in those tumors associated with an activated p53 oncogene. In contrast, wild type p53 polypeptide does not accumulate and is not easily detected. Because the wild-type p53 polypeptide is barely detectable in normal cells, due to its extremely short half-life, the presence of substantial amounts of p53 polypeptide establishes that the protein contains both a mutation resulting in extended half-life and the consequent phenotype of a dominant oncogenic protein. Stabilization of the mutant p53 polypeptide may be due to its ability to form complexes with other molecules such as heat shock proteins. Alternatively, stabilization of the mutant p53 polypeptide may be due to mutations in the primary sequence of the polypeptides which make them intrinsically more stable. Further alternatively, stabilization of the mutant p53 polypeptide may be due to post-translational modifications such as hyperphosphorylation (5-8, 27).
As previously discussed, the wild-type p53 oncogene is mutated at high frequency in the majority of human cancers. One of the first indications that p53 polypeptide expression may have been associated with some forms of human cancer was the observation that about 9% of patients with breast carcinoma (14 out of 155 samples tested) had auto-antibodies to p53 polypeptides (28). The occurrence of autologous antibodies directed against p53 polypeptides in patients with tumors indicated that the p53 polypeptide associated with the tumor was sufficiently altered so that it became immunogenic. It is important to note that at the time of such findings it was not possible to distinguish whether the auto-antibodies so observed were directed against wild-type or mutant p53 polypeptides (28). Indeed, the very existence of mutant p53 polypeptides had not been recognized at the time such auto-antibodies to p53 polypeptides were observed (28).
One early study found that tumors from 24% of breast cancer patients showed elevated levels of p53 polypeptide (17). An additional study showed that 40% of human breast cancer biopsies showed elevated levels of p53 polypeptides (18). Similarly, up to 55% of colon cancer tumor samples showed overexpression of p53 polypeptide based upon immunohistochemical data (19). None of these assays distinguished between wild-type and mutant p53 polypeptides.
In 1990, a more extensive study using the monoclonal antibody PAb240, which is selectively reactive with mutant p53 polypeptides, was performed. This study found that mutant p53 polypeptides could be detected in 50% of colon cancer, 30% of breast cancer and 70% of lung cancer tumor sections (4). However, mutant p53 polypeptides could not be detected in any normal or premalignant tissues from these patients. Other investigations have found overexpression of mutant p53 polypeptide in patients with leukemia or lymphoma (20, 21, 29). It is becoming increasingly apparent that when proper care is taken to preserve the specimen, a high percent of cancer biopsies examined are found to express elevated amounts of mutant p53 polypeptide.
Before applicants invention, histopathology had been the only technique which showed the correlation between mutant p53 polypeptides and neoplasia. The literature described monoclonal antibodies which specifically recognize and bind mutant p53 polypeptides, however, such antibodies did not (1) detect mutant p53 polypeptides in biological fluids or (2) imply that detection of mutant p53 polypeptide in biological fluids could effect the diagnosis of or monitor neoplastic conditions. Moreover, since mutant p53 polypeptides are located in the interior of the cell, one of ordinary skill in the art would not expect to detect mutant p53 polypeptides in substantially cell-free biological fluids. Thus, this invention is based upon the discovery that normally intracellular, mutant p53 polypeptides may be detected in biological fluids such as serum and that such detection may be utilized to diagnose or monitor neoplastic conditions or states. Accordingly, the establishment of a serum-based diagnostic marker for human cancer would have significant commercial applications. Using assay kits, blood drawn from patients could be routinely and easily assayed for mutant p53 polypeptide concentration. The correlation between the measured mutant p53 polypeptide concentration and the presence of neoplastic disease in the subject patient provides a means for early detection of the cancer. In addition, because of the ease for effecting the invention described herein a much larger segment of the population can be tested. Furthermore, the invention should have wide applicability in basic- and clinical- research applications.